Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent medical problem that affects approximately a third of the US population. Patatin-like phospholipase domain containing 3 (PNPLA3) was first implicated in the metabolism of hepatic triacylglycerides (TAGs) when our lab found that a missense mutation that substitutes isoleucine at position 148 with methionine (I148M) was associated with non-alcoholic fatty liver disease in humans. Homozygotes with the variant allele (148M) have a 2-fold higher risk of hepatic TAG accumulation than homozygotes with the wild type allele (148I). The function of PNPLA3 and the cause of I148M induced hepatic TAG accumulation remains unclear. Since fatty liver represents an accumulation of fatty acids in the form of triglycerides, I will adapt and/or develop targeted liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods to measure the concentrations of different classes of fatty acid-containing lipids in liver adipose tissue, and plasma from mouse models of PNPLA3-associated steatosis. Mice will be studied on both a normal chow diet and on diets designed to exacerbate or alleviate PNPLA3-I148M associated accumulation of hepatic TAG. I will then extend these LC-MS/MS-based methods to determine the flux of each lipid class by measuring the rate of incorporation of deuterium from deuterated water (D2O) in cultured cells that overexpress PNPLA3, and in genetically manipulated mouse models. Alternative stable isotope labeling strategies, such as 13C-glycerol or 13C-fatty acid incorporation, will also be employed to compliment the D2O incorporation data. Execution of this research proposal will provide training in the application of metabolomics technology to study human disease and shed insight into the function of PNPLA3 and its role in the progression of fatty liver disease.